Dendritic spines are small, actin-rich protrusions on the surface of neuronal dendrites that serve as sites of excitatory synaptic input and memory formation. Previous studies have implicated myosin II, the conventional non-muscle myosin, in spine organization and function. Here we have explored the localization within cerebellar Purkinje neurons (PNs) of myosin 18A (M18A)-alpha and M18A-beta, two spliced isoforms of M18A, a recently discovered member of the myosin super family that is similar to, and yet distinct from, class II myosins. These distinctions include a C-terminal PDZ-ligand motif in both spliced isoforms, and in the case of M18A-alpha, a 300-residue N-terminal extension that harbors an apparent ATP-independent actin-binding site, a Lys- and Glu-rich region (KE region), and a PDZ domain. The fact that M18A-beta lacks this N-terminal extension suggests distinct functions for these two isoforms. Using GFP-tagged versions of M18A-alpha and beta and a novel system for gene transduction, we find that M18A-alpha, but not M18A-beta, localizes dramatically to the dendritic spines of PNs in dissociated culture. Moreover, M18A-alphas spine distribution overlaps extensively with that of F-actin, as revealed using a novel reporter for F-actin (F-Tractin). The postsynaptic, spine localization of M18A-alpha was confirmed by immuno-EM of cerebellar tissue sections using a highly specific anti-M18A antibody. Importantly, M18A-alphas N-terminal extension by itself targets extensively to spines, arguing that it is largely if not entirely responsible for the myosins spine localization. Function-blocking point mutations show that the KE region and the PDZ domain within M18A-alphas N-terminal extension do not determine spine targeting. Rather, the interaction of the ATP-independent actin-binding site with actin filaments appears to drive the spine localization of M18A-alpha. Consistently, in vitro binding assays using M18A-alphas N-terminal extension show that it binds F-actin with moderately high affinity (305 105 nM). We suggest that M18A-alpha may play important roles in the development, organization, and/or function of PN spines. Future experiments using a conditional M18A knockout mouse, which we have successfully created, in combination with the PN-specific expression of Cre recombinase, will alow us to address the physiological significance of the myosin's spine localization in PNs.